Living organisms utilize carbohydrates as essential energy storage molecules. Starch is the predominant carbohydrate storage molecule in plants while glycogen is utilized in animals. Starch is a water-insoluble polymer that requires the concerted activity of kinases and phosphatases to solubilize the outer surface of the glucan and mediate starch catabolism. All known plant genomes encode the glucan phosphatase Starch Excess4 (SEX4). SEX4 can dephosphorylate both the starch granule surface and soluble phosphoglucans and is necessary for processive starch metabolism. The physical basis for the function of SEX4 as a glucan phosphatase is currently unclear. Herein, we report the crystal structure of SEX4, containing phosphatase, carbohydrate-binding, and C-terminal domains. The three domains of SEX4 fold into a compact structure with extensive interdomain interactions. The C-terminal domain of SEX4 integrally folds into the core of the phosphatase domain and is essential for its stability. The phosphatase and carbohydratebinding domains directly interact and position the phosphatase active site toward the carbohydrate-binding site in a single continuous pocket. Mutagenesis of the phosphatase domain residue F167, which forms the base of this pocket and bridges the two domains, selectively affects the ability of SEX4 to function as a glucan phosphatase. Together, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase.carbohydrate | Lafora disease | laforin | phosphorylation P lants and animals store carbohydrates as starch and glycogen, respectively. Starch is produced in diurnal cycles and is composed of <10% w∕w amylose and >80% w∕w amylopectin in Arabidopsis thaliana leaves (1). Amylose is a linear molecule composed of glucose moieties linked by α-1,4-glycosidic linkages with very few branches. Amylopectin, which is similar to glycogen, is composed of α-1,4-glycosidic linkages with α-1,6-glycosidic branches, but amylopectin branches are arranged in clusters at regular intervals and the branches form double helices that pack together to form crystalline lamellae (2, 3). The decreased branching and crystalline lamellae of amylopectin are key contributors to the insolubility of starch, while glycogen has more branches and is water-soluble.Starch is a water-insoluble polymer whose surface is inaccessible to most enzymes. Recent work convincingly demonstrates that reversible starch phosphorylation and dephosphorylation is essential for processive starch metabolism (reviewed in refs. 4-7). An essential signal triggering starch catabolism is phosphorylation on the C6 position of glucose moieties on the surface of starch by glucan water dikinase (GWD/R1) (8, 9). C6 phosphorylation triggers C3 phosphorylation by phosphoglucan water dikinase (PWD) (8,10,11). Recent data suggest that C6 phosphorylation fits within the unphosphorylated structure of the amylopectin helix, but C3 phosphorylation imposes significant steric effects and is predicted t...
Background: Lafora disease (LD) is a fatal autosomal recessive neurodegenerative disease. A hallmark of LD is cytoplasmic accumulation of insoluble glucans, called Lafora bodies (LBs). Mutations in the gene encoding the phosphatase laforin account for ~50% of LD cases, and this gene is conserved in all vertebrates. We recently demonstrated that laforin is the founding member of a unique class of phosphatases that dephosphorylate glucans.
Laforin and Starch Excess 4 (SEX4) are founding members of a class of phosphatases that dephosphorylate phosphoglucans. Each protein contains a carbohydrate binding module (CBM) and a dual specificity phosphatase (DSP) domain. The gene encoding laforin is mutated in a fatal neurodegenerative disease called Lafora disease (LD). In the absence of laforin function, insoluble glucans accumulate that are hyperphosphorylated and exhibit sparse branching. It is hypothesized that these accumulations trigger the neurodegeneration and premature death of LD patients. We recently demonstrated that laforin removes phosphate from phosphoglucans and hypothesized that this function inhibits insoluble glucan accumulation. Loss of SEX4 function in plants yields a similar cellular phenotype; cells accumulate an excess amount of insoluble, hyperphosphorylated glucans. While multiple groups have shown that these phosphatases dephosphorylate phosphoglucans, there is no structure of a glucan phosphatase and little is known about the mechanism whereby they perform this action. We utilized hydrogen-deuterium exchange mass spectrometry (DXMS) and structural modeling to probe the conformational and structural dynamics of the glucan phosphatase SEX4. We found that the enzyme does not undergo a global conformational change upon glucan binding, but instead undergoes minimal rearrangement upon binding. The CBM undergoes increased protection from deuteration when bound to glucans, confirming its role in glucan binding. More interestingly, we identified structural components of the DSP that also undergo increased protection from deuteration upon glucan addition. To determine the position of these regions, we generated a homology model of the SEX4 DSP. The homology model shows that all of these regions are adjacent the DSP active site. Therefore, our results suggest that these regions of the DSP participate in presenting the phosphoglucan to the active site and provide the first structural analysis and mode of action of this unique class of phosphatases.
We report on a pair of MSP (Mathematics & Science Partnership) START pilot projects designed to identify nanoscience experiments that will fit within the Alabama course of study for use in Alabama K-12 classrooms. As part of the first project we are testing the development, refinement and evaluation of an activity already partly developed. The form of this activity has had input from a focus group of RETs who were tasked to provide input into the activity and how it can be matched to components of the Alabama Course of Study. This activity consists of using sparks generated by abrasion of misch metal by sand paper of different grit size. Different grit sizes produce metal particles of different sizes, resulting in sparks of different size and length. If done in a dry box no sparks are produced and the powder left is not pyrophoric, demonstrating that high surface area, heat and oxygen are all required to produce sparks. SEM characterization of the powder allows the particle sizes to be determined, giving the correlation between size, grit size and spark track length. The activity was tested on groups of middle school science campers at McWane Science Center, and after evaluation, further modified to increase student interest and impact. The activity was then tested on grades 6-8 in a middle school classroom by a graduate student/undergraduate student team.
Reversible phosphorylation modulates many proteins involved in glycogenesis and glycogenolysis. Recent results from mammalian cells, mouse models, eukaryotic algae, and plants have identified a new phosphorylation target in glycogen metabolism, the glucan itself.Mutations in EPM2A lead to Lafora disease, a fatal neurodegenerative disease. EPM2A encodes laforin, a bimodular protein with an amino‐terminal carbohydrate binding module and a carboxy‐terminal dual specificity phosphatase domain. We recently demonstrated that laforin removes phosphate from phosphoglucans.Starch Excess 4 (SEX4) encodes a similar phosphatase in plants, and we demonstrated that laforin and SEX4 are the founding members of the glucan phosphatases. Multiple groups have now shown that these phosphatases dephosphorylate phosphoglucans, but the physical basis for the function of laforin and SEX4 is unclear.We determined the crystal structure of SEX4, and found that it contains a phosphatase domain (DSP), carbohydrate binding module (CBM), and a previously unknown carboxy‐terminal domain. Hydrogen‐deuterium exchange mass spectrometry (DXMS) identified regions of the DSP domain that interact with glucans. Cumulatively, these results reveal the unique tertiary architecture of SEX4 that provides the physical basis for its function as a glucan phosphatase and present a model for the function of laforin.
Background: Longitudinal research on COVID-19 impacts on drinking is scarce and largely restricted to comparing drinking levels before and after the introduction of COVID mitigation measures. This brief snapshot of behavior ignores the extended pre-COVID drinking trajectory, which may be decreasing increasing, or remaining stable over time. Behavioral economics predicts that pandemic-related constraints on behavioral alternatives to alcohol and drug use, and decreased constraints on alcohol, may result in increases in drinking at later stages of the pandemic. Therefore, the current study characterized drinking trajectories among emerging adults before and during the pandemic and investigated time-invariant demographic predictors and time-varying behavioral economic predictors of trajectories of drinking and behavioral economic variables. Methods: A pandemic-focused survey was distributed between May 15 and June 29, 2020 to emerging adults participating in an ongoing longitudinal study involving pre-COVID data collection every four months. Participants with four pre-COVID assessments were included in the current study (N = 312, ages 21.5-24 years; 65.1% female). Results: Linear piecewise models best fit the drinking days and drinks per week data, suggesting a pandemic-related disruption of ongoing drinking trajectories. After controlling for all other time-invariant predictors, lower environmental reward was associated with greater increases in heavy drinking days and income loss was associated with lower drinking days, drinks, and heavy drinking days per week. In parallel LGCM models, increases in alcohol demand indices were generally associated with increases in drinking from the pre- to the post-COVID onset timepoint. Conclusions: The results suggest that the pandemic attenuated ongoing declines in drinking trajectories and highlight the value of examining trajectories to characterize COVID-19-related effects. Behavioral economic measures of environmental and alcohol reward may be useful predictors of changing alcohol use patterns, particularly in the context of emergent public health crises.
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